The myotendinous junction (MTJ) is the primary site for force transmission from the interior of the muscle cell, across its membrane, and to the extracellular matrix (ECM). In healthy muscle tissue, the MTJ provides resistance against the mechanical stress generated during muscle contraction, and it is now known that any decrease in MTJ stability leads to muscle detachment in diverse organisms. Most significantly, it is this detachment phenotype that typifies a series of congenital, progressive myopathies in humans. While many features concerning MTJ formation, structure, and function are conserved between both vertebrates and invertebrates, studies in the genetically tractable organism Drosophila melanogaster have proven instrumental in uncovering many proteins essential for MTJ assembly and function and muscle development as a whole. Therefore, the overall goal of this application is to use the fly model to better understand MTJ formation and how defects in MTJ stability may lead to the onset and progression of myopathies. The evolutionarily conserved Elmo-Myoblast city (Mbc) complex activates the small GTPase Rac during Drosophila muscle development. While the primary role of Rac lies in regulation of the actin cytoskeleton, other signaling components that function in Elmo-mediated myogensis - including the signals that initiate and regulate Elmo- Mbc activity - have remained elusive. This proposal expands upon preliminary data which show that (i) Elmo is also required for proper muscle-tendon attachment in the fly, and that (ii) there exist two new Elmo-binding proteins, both of which are required for muscle attachment at the MTJ. The role(s) of these Elmo-containing complexes will be examined using the mature Drosophila MTJ as a model for both muscle-tendon signaling and subsequent force transmission generated upon muscle contraction. To test our overall hypothesis that these new Elmo protein complexes function to mediate cytoskeletal rearrangement during Drosophila muscle attachment, we will use a powerful combination of genetic, biochemical, and imaging approaches to pursue the following specific aims: (1) dissect the role of Elmo in MTJ formation and/or Rac activation; (2) identify the mechanism by which Elmo and associated proteins function to maintain stable MTJs; and (3) understand the function of Elmo complexes in mitochondrial localization during muscle attachment. PUBLIC HEALTH RELEVANCE: Defects in the formation and/or function of stable muscle attachments are implicated in congenital human myopathies, and the progressive muscle weakness resulting from these myopathies is an obvious detriment to human health. The functional conservation of proteins required for muscle development across diverse species is well-established. Thus, we will use the well-established genetics in the fly to both define the roles of new proteins that contribute to muscle-tendon formation and function.